The present invention relates generally to the field of ligation of nucleic acids, using phosphorothioate derivatives as a means of ligating single stranded oligonucleotides, and where such oligonucleotides contain a point mutation, as a means of detecting single nucleotide polymorphisms.
The ability to detect single base differences in DNA is of great importance in molecular genetics. Specific identification of point mutations is playing an increasingly important role in diagnosis of hereditary disease and in identification of mutations associated with drug resistance. Because of the high fidelity of ligation, enzymatic ligation methods have proven useful in a number of novel gene detection techniques.
Polynucleotide ligases are ubiquitous cell proteins that are required for a number of important cellular processes, including replication, repair and recombination of DNA. One of the best-characterized enzymes that joins DNA ends is T4 DNA ligase, first isolated some three-decades ago (for reviews see 1-3). This and related proteins catalyze ATP-dependent phosphodiester bond formation between the 5xe2x80x2-phosphate and 3xe2x80x2-hydroxyl groups of adjacent DNA strands (4,5). Duplex DNA molecules with either cohesive ends or blunt ends can serve as ligation substrates (6,7). T4 DNA ligase can also repair nicked DNA duplexes efficiently and thus the enzyme is often used for joining two DNA segments that are hybridized adjacent to each other on a complementary strand.
The use of circular DNAs (which can be formed by ligation, internal or external, of single stranded oligonucleotides) in such methods of amplification as rolling circle amplification technology (RCAT(trademark)) promises to greatly improve the performance of gene-based diagnostic testing and to facilitate the detection of a wide variety of infectious agents, cancerous cells, and genetic variations (also called polymorphisms) (71). Since the discovery of circular DNA to serve as a template for DNA polymerases (72), there has been increasing demand for the synthesis of circular oligonucleotides. Although there have been reports of successful enzymatic ligation to produce circular DNA, the yields of circles (less than 100 nucleotides or 100 nt) have been modest. Non-enzymatic ligation strategies have been somewhat more successful in the synthesis of small circular DNAs (less then 50 nt) on solid supports. Several approaches using non-enzymatic methods have recently been described. However, synthesis of circular DNA (larger than 50 to 100 nt or nucleotides) by non-enzymatic methods is a slow, entropically disfavored process and remains a synthetic challenge.
Kool et al have reported a method of circle synthesis (See Kool, E. T. et. al, Nature Biotechnol (2001)19, pp. 148-152; Kool, E. T. et. al., Nucleic Acids Res, (1995)I. 23 (17), pp. 3547-355; Kool, E. T. et.al (1999) 27(0.3), pp 875-871. However, Kool""s published report uses a chemical ligation approach wherein the ligation reaction produces a DNA containing sulfur in the bridging junction as 5xe2x80x2-S-thioester linkage at the site of ligation. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5xe2x80x2-iodide with 3xe2x80x2-phosphorothioate group (see, in general, the cited references herein). In addition, Kool et. al has reported a reagent free autoligation approach where 5xe2x80x2-S-thioester bonds formation between 3xe2x80x2-phosphorothioate and a 5xe2x80x2-iodide, which acts as a leaving group.
Other published methods report a chemical ligation approach for the synthesis of single stranded circular DNA wherein the modified circular DNA comprises a single 5xe2x80x2-S-thioester linkage with sulfur located in the 5xe2x80x2-bridging region. Other strategies have used sulfur atoms to replace specific non-bridging phosphate oxygens in RNA. Eckstein, F, Angew. Chem. Intl. Ed. Engl. 22, pages 423 (1993) have synthesized phosphorothioate-linked polyribonucleotides as early as 1967 using DNA-dependent RNA polymerase from E.coli. Several other polymerases proved useful in the synthesis of the phosphorothioate linked ribo and deoxy oligonucleotides.
When a single base pair mismatch exists at either side of the ligation junction, the efficiency of the enzyme in ligating the two oligonucleotides decreases markedly. This high sequence selectivity has resulted in the development of novel sequence detection methods using this enzyme. These approaches include the ligase detection reaction (LDR) (8-10) and the ligase amplification reaction (LAR) (11). T4 DNA ligase displays selectivity against single base mismatches on the order of 2 to 6 fold in yield and conditions such as the presence of spermidine, high salt and low enzyme concentration have been reported to improve the fidelity of ligation to as high as 40 to 60-fold (9, 10). The thermostable DNA ligase from Thermus thermophilus (Tth DNA ligase) has been reported to have higher fidelity (mismatch discrimination of 450 to 1500-fold in rate) and has been measured for the wild-type enzyme with optimized mismatch location at the 5xe2x80x2-side of junction (12,13), making its use preferable in some sequence detection methods including the ligase chain reaction (14-16).
Since the discovery of ligases there have also been developed a number of non-enzymatic approaches to joining the ends of two DNA strands (17-29). These chemical ligations have been achieved via oxidative coupling of terminal 3xe2x80x2-phosphorothioates and displacement of 5xe2x80x2-bromide from a monoacetylamino bromide (19), 5xe2x80x2-tosylate (17) and a 5xe2x80x2-iodide (30). The resulting DNA contains the modified 5xe2x80x2-S-thioester linkage in the bridging position. Strategically placed sulfur atoms in the backbone of nucleic acids have found widespread utility in probing of specific interactions of proteins, enzymes and metals. Sulfur replacement for oxygen has also been carried out at the 2xe2x80x2-position of RNA (37-39) and in the 3xe2x80x2-5xe2x80x2-positions of RNA (40-45) and of DNA (46-56) Polyribonucleotide containing phosphorothioate linkages were obtained as early as 1967 by Eckstein et al. using DNA-dependent RNA polymerase from E.coli (57). DNA-dependent RNA polymerase is a complex enzyme whose essential function is to transcribe the base sequence in a segment of DNA into a complementary base sequence of a messenger RNA molecule. Nucleoside triphosphates are the substrates that serve as the nucleotide units in RNA. In the polymerization of triphosphates, the enzyme requires a DNA segment that serves as a template for the base sequence in the newly synthesized RNA. In the original procedure, Uridine 5xe2x80x2-O-(1-thiotriphosphate), adenosine 5xe2x80x2-O-triphosphate, and only d (AT) as a template was used. As a result, an alternating copolymer [Ap (S) UpAp (S) Up] is obtained, in which every other phosphate was replaced by a phosphorothioate group. Using the same approach and uridine 5xe2x80x2-O-(1-thiotriphosphate) and adenosine 5xe2x80x2-O-(1-thiotriphosphate), polyribonucleotide containing an all phosphorothioate backbone was also synthesized (58). In both cases, nucleoside 5xe2x80x2-O-(1-thiotriphosphates) as a mixture of two diastereomers were used.
The stereo chemical course of polymerization catalyzed by Escherichia coli polymerase was determined by Eckstein et al using the 1-thio-analog of adenosine 5xe2x80x2-O-triphosphate as a substrate (58,59). The Sp isomer was found to serve as an enzyme substrate and was further used with Uridine 5xe2x80x2-O-triphosphate and a DNA template of poly-d (AT). The resulting RNA polymer was the complementary alternating copolymer of adenosine and uridine linked by alternating 3xe2x80x2-5xe2x80x2-phosphodiester and 3xe2x80x2-5xe2x80x2-phosphorothioate bonds. Other polymerases useful in the synthesis of the phosphorothioate backbone bearing polyribonucleotides and polyrdeoxyibonucleotides have been described (60-68).
In addition, Single Nucleotide Polymorphisms (SNPs) have been characterized using a variety of methods. Such methods include the direct or indirect sequencing of the site, the use of restriction enzymes where the respective alleles of the site create or destroy a restriction site, the use of allele specific hybridization probes, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism, or by other biochemical interpretation. However, no assay yet exists that is both highly accurate and easy to perform.
Oligonucleotide ligation assay (OLA) is an assay proposed for SNP determination [Landergen et al., Science (1988) 241:1077-1080]. The OLA protocol uses two oligonucleotides, which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleeotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. OLA is capable of detecting point mutations [Nickerson, D. A. et. al., Proc. Natl. Acad. Sci. U.S.A., (1990) 87:8923-8927]. In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. Assays, such as OLA, require each candidate dNTP of a polymorphism be separately examined, using a separate set of oligonucleotides for each dNTP. The major drawback is that ligation is not a highly discriminating process and non-specific signals can be a significant problem.
Since phosphorothioates exist as diastereomers, automated synthesis of DNA on solid phase synthesizers results in mixtures of Rp and Sp diastereomers at the individual phosphorothioate linkages. Replacement of oxygen with a sulfur atom in 5xe2x80x2-phosphate of DNA results in increased bond length, reduced electronegativity and increased bond length. In addition, sulfur substitution for oxygen should also result in reduced hydration and altered metal ion affinity. Hence, these physico-chemical changes could have significant effect on any of the three fundamental steps in enzymatic ligation.
The major advantages of the present invention over other methods, including the autoligation approach, include the following: the autoligation approach produces a single 5xe2x80x2-S-thioester linkage where the sulfur is located in the 5xe2x80x2-bridging region whereas the procedures of the present invention do notxe2x80x94the sulfur is outside of the bridge so that the 5xe2x80x2-thio phosphate directed ligation as disclosed herein produces a circular DNA with sulfur being located in the non-bridging region of phosphorothioate at 5xe2x80x2-end of the incipient circle; prior art approaches include the reagent free autoligation method whereas the present invention is enzyme based and provides great sensitivity to the presence of nucleotide mismatches; and the sulfur atom introduced in the processes of the invention resides in the non-bridging position which is isosteric to the naturally occurring phosphate linkage and therefore does not interfere with subsequent uses of the circular product (such as subsequent rolling circle amplification). In sum, the current method is diastereo-selective approach and has great potential for allele discrimination.
In one aspect, the present invention relates to a method for the ligation of nucleic acids that is at once efficient, low cost and amenable for large-scale ligation of DNA and analogs thereof, for use as probes, diagnostic agents, and therapeutic agents.
It is one object of the present invention to provide closed circles formed by the processes of the invention wherein said circles contain a phosphorothioate linkage but are useful in many, if not all, of the same processes for which conventional single-stranded closed circles find use because the circles contain phosphodiester bonds wherein the sulfur atom is not part of the bridging atoms.
It is another object of the present invention to provide a process for rolling circle amplification (RCA) employing as templates the single stranded circles formed by the methods of the invention and thereby amplifying selected nucleotide sequences.
In another aspect, the present invention relates to a process for detecting nucleotide mismatches, such as in single nucleotide polymorphisms (SNPs), comprising:
contacting a first oligonucleotide, comprising first and second segments, with a second oligonucleotide wherein said second oligonucleotide comprises a first complementary segment, a second complementary segment, and a third segment and wherein said second oligonucleotide also comprises a phosphorothioate at its 5xe2x80x2-terminus,
and wherein said first and second complementary segments of said second oligonucleotide are complementary to the first and second segments of said first oligonucleotide one of which first oligonucleotide segments comprises at least one mismatched nucleotide, and wherein said third segment is not complementary to said first oligonucleotide,
and wherein said contacting occurs under conditions promoting hybridization of said first and second segments with said first and second complementary segments, and wherein said hybridization results in the formation of a complex in which the 5xe2x80x2- and 3xe2x80x2-ends of said second oligonucleotide are adjacent to each other, and said 3xe2x80x2-nucleotide may be opposite a mismatched nucleotide of said first oligonucleotide,
contacting said hybridized complex with a ligation catalyst under conditions promoting ligation of the 5xe2x80x2- and 3xe2x80x2-ends of said second oligonucleotide in said hybridized complex when said mismatch is not present, and
detecting formation of ligated second oligonucleotide wherein reduced or non-formation thereof indicates the presence of a nucleotide mismatch.
In a preferred embodiment of this process, the catalyst is an enzyme, most preferably Ampligase or T4 DNA or RNA ligase. For RNA ligations, see Faruqi, U.S. application Ser. No. 09/547,757, filed Apr. 12, 2000, the disclosure of which is hereby incorporated by reference in its entirety.
In another aspect, the present invention relates to a detection process for single nucleotide polymorphisms as described herein but further comprising detecting the occurrence of ligated second oligonucleotide by contacting the third segment of ligated second oligonucleotide with a primer oligonucleotide complementary to said third segment under conditions promoting hybridization of said primer to said third segment and further contacting said complex with a rolling circle amplification (RCA) enzyme under conditions promoting rolling circle amplification of said ligated second oligonucleotide. Here, the first oligonucleotide could be derived from genomic DNA.